PROJECT SUMMARY The aquatic bacterium Vibrio cholerae causes human diarrheal disease that can range from mild to deadly. Pandemic O1/O139 strains cause cholera, a potentially deadly disease, whereas non-O1/O139 strains, also known as ?environmental? V. cholerae, cause a range of diarrheal diseases from mild to severe. Animal models have been useful in characterizing major V. cholerae virulence factors, but rabbits and mice are not natural V. cholerae hosts and require absent or damaged microbiota to enable colonization. Vertebrate fish are natural V. cholerae hosts and recent work has established zebrafish as a model that recapitulates the entire infectious process in the presence of intact intestinal microbiota. Fish are rapidly colonized by V. cholerae exposure in water, develop diarrhea, excrete large numbers of V. cholerae, and transmit the infection to nave fish. Because this is a model in a natural host, it presents new opportunities for V. cholerae study that are not possible with mammalian models. However, the V. cholerae factors that act in fish pathogenesis are unknown for both O1 and non-O1/O1319 strains. This proposal will examine the infectious processes used by both O1 and non-O1/O139 V. cholerae and test the following hypotheses: 1) V. cholerae produce specific colonization factors in fish that are also important in human infections. These factors are shared among all V. cholerae strains. This hypothesis will be tested using Tn-Seq to identify genes important for fish colonization. 2) V. cholerae produces factor(s) that directly cause diarrhea. This hypothesis will be tested by knocking out known toxins in each strain and measuring diarrhea. 3) V. cholerae perturbs specific components of the intestinal microbiota, creating a niche for colonization. This hypothesis will be tested by assessing microbiome before and after infection. 4) The type six secretion system (T6SS) is important for competition with intestinal microbiota. This hypothesis will be tested by knocking out T6SS and further identifying T6SS effectors that are important for this competition. 5) V. cholerae excreted by fish are hyperinfectious, as they are in human cholera stool, and this is a likely source of V. cholerae outbreaks. This hypothesis will be tested by comparing infectious doses required for colonization from in vitro grown and fish-passaged V. cholerae. 6) V. cholerae shifts its gene expression program prior to host escape to enable transmission and contribute to hyperinfectivity. This hypothesis will be tested using RNA-Seq to determine global changes in gene expression between in vitro grown, actively colonizing, and excreted V. cholerae. Completion of the proposed work, using zebrafish as a natural V. cholerae host model, will significantly advance our understanding of the environmental lifestyle of V. cholerae in a natural reservoir, the requirements for V. cholerae to become a pathogen, and should uncover new targets for therapeutics, environmental remediation, and disease prevention. The long term goal of this project is to use the zebrafish model to better understand V. cholerae pathogenesis in humans and identify new strategies to combat V. cholerae disease and transmission. .